1. Field of the Invention
This invention relates to compositions of matter, pharmaceutical compounds, methods of synthesizing such compounds and methods for using such compounds to treat animals infected with a pathogenic microorganisms, specifically mycobacteria. The invention relates in particular to compositions of matter and pharmaceutical compositions thereof for the treatment of tuberculosis and other Mycobacterium-caused diseases.
2. Background of the Related Art
Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. This disease typically arises after inhalation in phagocytic macrophages in the lung, where characteristic localized sites of infection (termed tubercules) are formed and comprise sites of further systemic infection. Although previously well-controlled by antibiotics such as isoniazid, the development of drug-resistance by the infectious agent, and the increased numbers of immune-compromised individuals being affected by the AIDS crisis has created a near epidemic of tuberculosis cases world-wide. In 1997, the World Health Organization reported tuberculosis to be the world""s top infectious killer.
About one-third of new tuberculosis cases are resistant to the current drug-treatment regimes. It is estimated that drug-resistant tuberculosis accounts for between 2% and 14% of total tuberculosis cases worldwide. As tuberculosis is spread by air-borne droplets from coughing by infected individuals, and its spread is further facilitated in crowded environments such as cities, there is a great potential for a precipitous increase in tuberculosis infections which will not be easily controlled by conventional medicinal intervention, such as isoniazid administration. Lethal strains of tuberculosis have the potential for rapid spread, since only about one in ten patients receives the medical treatment necessary to contain and successfully treat the disease. Thus, there exists in this art a need to develop new and better treatments for tuberculosis, particularly tuberculosis infections resistant to traditional antibiotic treatments.
There is also a need in the art for more effective anti-tuberculosis drugs to which M. tuberculosis is not resistant and, most advantageously, drugs having a low resistance development potential.
In addition, there are a number of other human and animal diseases caused by mycobacteria, including for example leprosy (Hansen""s disease), lymphadenitis, a variety of pulmonary and skin diseases, and wound infection. Although less prevalent, each of these diseases is associated with morbidity, mortality and economic costs such as lost production time and the cost of medical treatment. Resistance to drugs used heretofore to control and treat such diseases is also a current problem, thus raising a further need in this art for more effective drugs against many different Mycobacterium species.
The present invention is directed to improved antibiotic compounds, specifically pharmaceutical compositions thereof, and methods for producing and administering such pharmaceutical compositions, for treatment of diseases having a Mycobacterium etiology. In particular, the invention is directed towards delivery of antimicrobial compounds, drugs and agents specific for treatment of tuberculosis and other Mycobacterium-caused diseases in humans.
The invention provides improved antimycobacterial drugs that are xe2x80x9cactivatedxe2x80x9d embodiments (as defined herein) of competitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d inhibitors of long chain enol-acyl carrier protein reductase (InhA), a Mycobacterium-specific enzyme necessary for the production of mycolic acid, which an essential component of the mycobacterial cell wall. Inhibition of this enzyme by isoniazid is the basis of current anti-tuberculosis treatment modalities, and resistance to isoniazid is the principle form of drug resistance exhibited by mycobacteria. The compounds of the invention overcome resistance by being xe2x80x9cpre-activatedxe2x80x9d, i.e., these compounds do not rely on activation in the mycobacterium-infected cell for activity (unlike isoniazid itself). Thus, it is expected that resistance is less likely to be developed against these drugs. In a preferred embodiment, these compounds have the generic structure: 
wherein X can be C or O; Y can be N or C; R1 and R2 can each be independently an electron pair, H, CH3, CH2xe2x80x94CH3, or O(CH2)3O or together can be xe2x95x90O, xe2x95x90CH2, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x95x90CHxe2x80x94CHxe2x95x90CH2, xe2x95x90CHxe2x80x94COOCH2xe2x80x94CH3, or OCH2.
Each of the compounds disclosed herein is an analog for nicotinamide adenine dinucleotide, a cellular component that mediates transfer of electrons in a number of cellular systems (including glycolysis, mitochondrial oxidative phosphorylation, fatty acid synthesis and breakdown, and other synthetic and metabolic pathways). It is known in the art that isoniazid, the traditional drug of choice for treating tuberculosis, is activated by a M. tuberculosis produced catalase/peroxidase (as shown in FIG. 1); Quemard et al., 1996, J. Amer. Chem. Soc. 118: 1561; Sacchettini and Blanchard, 1996, Res. Microbiol. 147: 36; Zabinski and Blanchard, 1997, J. Amer. Chem. Soc. 119: 2331) to form an adduct with NAD (the resulting activated form of isoniazid is termed isoniazid-NAD analogue (INA); Rozwarski et al., 1998, Science 279: 98-102). A major route for isoniazid resistance to M. tuberculosis is mutation or inactivation of the catalase/peroxidase that converts isoniazid to INA, suggesting that mycobacteria may be less likely to develop resistance to INA that to isoniazid itself.
A major drawback for using INA directly to treat M. tuberculosis infections is that INA is expected to inhibit a variety of NAD-dependent host enzymes, with toxic or at least deleterious effects. In one embodiment of the invention, this limitation is overcome by providing alternative embodiments of INA that have been derivatized at one or more positions on the molecule required for enzyme binding. Both the formamide group of NAD and the adenine portion thereof bind to amino acid sequences in the NAD binding portion of NAD requiring enzymes that have been highly conserved in evolution (Rossman et al., 1978, Molec. Cell. Biochem. 21: 161-182; Baker et al., 1992, J. Molec. Biol. 228: 662-671; Zeng et al., 1995, Biochem. J. 310: 507-516). Modification of INA at either position disrupts binding to NAD-requiring enzymes.
In order to specifically target M. tuberculosis and other Mycobacterium-infected cells with an activated form of INA, most preferably phagocytic cells known to be in vivo reservoirs of Mycobacterium infection, inactivating modifications thereof are made using derivatizing groups that are specifically cleaved in Mycobacterium-infected cells. In a preferred embodiment, the derivatizing group is a urea moiety, because Mycoabacteria produce urease in infected cells that can cleave the urea group from the derivatized INA analogue and thus activate INA in such cells (Wayne and Kuica, 1986, BERGEY""S MANUAL OF SYSTEMIC BACTERIOLOGY (Sneath et al., eds.), Williams and Wilkins; Good et al., 1985, Ann. Rev. Microbiol. 39: 347). Preferred positions for derivatizing INA with said urea moieties include the formamide group of the pyridine portion of the NAD component of INA, and the 1-amino group of the adenine portion of NAD component of INA. Because mammalian cells not infected with Mycobacteria do not produce urease, the modified INA compounds of the invention do not have toxic or deleterious effects on such cells.
Particularly preferred targets of the pharmaceutical compositions of the invention are phagocytic cells, preferably macrophages and phagocytic neutrophiles and most preferably macrophages, mononuclear cells and phagocytic neutrophiles from lung tissue that are infected with M. tuberculosis, M. africanum, M. bovis or any other microorganism that causes tuberculosis in an animal, most preferably a human. Also preferred targets are cells infected with M. leprae, M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. xenopi, M. marinum, M. ulcerans, M. fortuitum and M. chelonae. 
The anti-mycobacterial compounds of the invention are advantageous because, inter alia, the compounds are xe2x80x9cactivatedxe2x80x9d inhibitors of a target enzyme specific for mycobacterial cells. Inhibition of this enzyme is unlikely to be disadvantageous to infected animals, since there is no eukaryotic or vertebrate analogue of this enzyme. In addition, the anti-mycobacterial compounds and pharmaceutical compositions thereof are provided in a prodrug form that is inactive in uninfected cells but is specifically activated in cells, most preferably phagocytic cells, infected to Mycobacteria species.
The invention provides a method of killing a microorganism infecting a mammalian cell, preferably a phagocytic mammalian cell. This method comprises contacting an infected phagocytic mammalian cell with the compositions of matter or pharmaceutical compositions of the invention in vivo or in vitro. The invention also provides methods for treating microbial infections in an animal, most preferably a human wherein the infecting microbe is present inside a phagocytic cell in the human, the method comprising administering a therapeutically effective amount of the compositions of matter or pharmaceutical compositions of the invention to the human in a pharmaceutically acceptable carrier. Thus, the invention also provides pharmaceutical compositions comprising the compositions of matter of the invention in a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition is formulated in an orally-administered dose. In most preferred embodiments, the infecting microorganism is a tuberculosis-causing microorganism such as M. tuberculosis, M. africanum or M. bovis. 
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.